CN107991766A - A kind of microscope and imaging method with three-dimensional imaging ability - Google Patents

Abstract

The invention discloses a microscope with three-dimensional imaging capability and a microscopic method thereof. The microscope comprises: at least one excitation device, which is used to generate a detectable contrast in the area of a sample to be measured along the direction of the excitation axis; at least one detection The device is used to detect the contrast produced in the region of the sample to be measured along the direction of the main axis of detection; at least one moving mechanism is used to generate relative motion between the sample and the excitation device and the detection device; wherein, the direction of the relative motion is the same as the The direction of the excitation principal axis is neither parallel nor perpendicular; the direction of the relative motion is neither parallel nor perpendicular to the direction of the detection principal axis. The invention can reduce the imaging pause in three-dimensional imaging, including uninterrupted imaging while the sample is moving, reduce the field of view conversion that must interrupt imaging, eliminate the blur caused by the sample movement during imaging, and improve the aberration caused.

Description

A microscope and imaging method with three-dimensional imaging capability

technical field

The invention relates to an imaging microscope and a microscopic imaging method, in particular to a high-throughput three-dimensional digital imaging microscope and a high-throughput three-dimensional digital microscopic imaging method with biology and medicine as the main application fields.

Background technique

Three-dimensional digital imaging is one of the important contents of modern microscopic imaging. Non-destructive imaging of more samples without reducing resolution is an important pursuit of 3D microscopic imaging methods.

In general, three-dimensional digital imaging probes the sample (excitation) in a way that produces a contrast response (such as fluorescence), records these contrasts in a certain way (photoelectric conversion and digitization), and uses each individual unit in the area to be measured The contrast is digitized into voxels as the final output. Therefore, the number of voxels, that is, the volume of the area to be measured in the sample divided by the volume corresponding to the voxels (determined by the required resolution), and the digitization speed determine an upper limit of the three-dimensional digital imaging speed. However, the imaging speed of existing technologies is far from reaching this limit. Taking the digital (16bit) speed of 400 megapixels per second as an example of the existing mainstream imaging equipment to image a mouse brain of 0.5 cubic centimeters, the three-dimensional volume pixel corresponds to the digital imaging of 1×1×1 micron unit (subcellular resolution) The upper limit of the speed should be 1250 seconds, which is about 21 minutes; the voxel corresponds to 5×5×5 micron units (cell body resolution) and it only takes 10 seconds. In the prior art, the representative speed of mouse brain imaging with submicron resolution is 3 days (Gong, H. et al. Nat. Commun. 7: 12142, 2016), and the latest result of cell body resolution is about 2 hours (Li Ye, et al. al., Cell, 165(7), 16 June 2016,). With existing technologies, the speed of excitation and recording is not the bottleneck. An important reason for this gap is that the existing technology cannot avoid imaging interruption during the imaging process, thereby shortening the effective imaging time.

Existing imaging technologies usually adopt such a scheme: first, image a plane perpendicular to the imaging direction (that is, the direction of the detection axis, usually referred to as the z direction), which can be point-by-point imaging or simultaneous imaging of part or the entire field of view to scan Confocal (scanning confocal) or optical section (SPIM, sometimes abbreviated as LSM) imaging is taken as an example; in the z direction, relative motion is performed according to the resolution requirement to reach a new plane, and this process takes about 10 milliseconds; Planar imaging; repeat the above movement and imaging until the sample is covered in the z direction, complete a three-dimensional imaging in a field of view, and then move to a new field of view in the direction perpendicular to z, this process takes up to hundreds of milliseconds; The above movement and imaging are repeated until the sample is also covered in the direction perpendicular to z to complete the three-dimensional imaging of the sample. The above process includes a large number of interrupted imaging processes. Each movement itself takes time, and it is necessary to wait for the vibration dissipation caused by the start and stop to continue imaging, resulting in long-term imaging interruption. In the case of a large sample volume and a high resolution requirement, the number of interrupted imaging increases cubically, which seriously affects the imaging efficiency. Some technologies reduce the interrupted imaging time with solutions such as electric focusing lenses, but they are only applicable to the z direction, and the scale is very limited, and the throughput of 3D digital imaging is only partially improved.

Fig. 1A to Fig. 1G show the scheme commonly used in the prior art to realize three-dimensional imaging: first, as shown in Fig. 1A, image a plane perpendicular to the imaging direction (that is, the detection axis direction, usually referred to as the z direction)—it can be Point-by-point imaging or simultaneous imaging of part or the entire field of view, taking scanning confocal (scanning confocal) or optical slice (SPIM, sometimes abbreviated as LSM) imaging as an example; relative movement in the z direction according to the resolution requirements to reach a new The imaging position is shown in Figure 1B; imaging at the imaging position is shown in Figure 1C; repeat the above movement and imaging, as shown in Figure 1D, until the sample is covered in the z direction, and a three-dimensional imaging in a field of view is completed; Move to a new field of view in the direction of z, as shown in Figure 1E; the three-dimensional imaging in the field of view is shown in Figure 1F; repeat the above movement and imaging until the sample is also covered in the direction perpendicular to z, and the three-dimensional imaging of the sample is completed as shown in Figure 1 1G shown. The above process includes a large number of interrupted imaging processes. Each movement takes time, and the vibration caused by the start and pause must be waited for to continue imaging, resulting in long-term imaging interruption. We can see that the aforementioned interrupted imaging is associated with movement. In the 3D imaging technology that combines multiple local imaging into overall imaging, certain forms of relative movement between the sample and the probe are unavoidable, but not all relative movements require interruption of imaging. The relative motion contained in traditional 3D imaging can be divided into two categories: the first type of motion: the imaging of different surfaces in the process of 3D imaging in a single field of view, and the small relative movement between each data reading, usually less than a few microns; The second type of movement: large relative movement that occurs during the conversion of the field of view, usually more than 100 microns.

references:

Gong, H. et al. High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level. Nat. Commun. 7: 12142, doi: 10.1038/ncomms12142 (2016).

Li Ye, et al. Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences, Cell, Volume 165, Issue 7, 16 June 2016, Pages1776-1788, ISSN 0092-8674, http://dx.doi.org/10.1016/j .cell.2016.05.010.

Contents of the invention

(1) Technical problems to be solved

The technical problem mainly solved by the present invention is to provide a three-dimensional digital imaging microscope and a three-dimensional digital microscopic imaging method, which can improve the effective imaging time and realize high-throughput three-dimensional digital microscopic imaging.

(2) Technical solutions

In order to solve the above technical problems, according to one aspect of the present invention, a microscope with three-dimensional imaging capability is provided, including: at least one excitation device, used to generate a detectable contrast in the sample area to be measured along the direction of the excitation axis; at least A detection device is used to detect the contrast produced in the region of the sample to be measured along the direction of the detection axis; at least one moving mechanism is used to generate relative motion between the sample and the excitation device and the detection device; wherein, the direction of the relative motion The direction of the relative motion is neither parallel nor perpendicular to the direction of the main axis of excitation; the direction of the relative motion is neither parallel nor perpendicular to the direction of the main axis of detection. In the overall process of imaging a three-dimensional region of the sample, both the imaging and the relative motion have the characteristics of non-stop, and the direction of the relative motion is neither parallel nor perpendicular to the direction of the excitation principal axis and the detection principal axis. Here, the choice regarding the direction of relative motion helps to reduce field of view transitions that must interrupt imaging.

Since the relative movement during imaging has the characteristics of uniform speed and basically no pause, the irregular vibration caused by speed change or starting and stopping is eliminated. The blurring caused by motion that may still exist is simple and consistent, and can be easily eliminated by deconvolution and other methods.

Preferably, in the effective detection area of the detection device, the sample unit corresponding to each imaged voxel is only excited by the excitation device for no longer than a specific time, during which the relative motion produces The displacement is not greater than the resolution requirement of the microscope. In this way, the relative motion produces negligible motion blur.

Preferably, the environment where the sample is located is filled with a transparent substance, and the refractive index of the transparent substance is approximately equal to that of the sample at that time.

Preferably, the excitation device excites the light source with a combination of one or several light emitting diodes, continuous lasers, and pulsed lasers.

Preferably, the detectable contrast is one or a combination of fluorescence, elastic scattered light, Raman scattering, SHG, THG, stimulated Raman scattering and other signals.

Preferably, the excitation device excites the sample by means of light sheet illumination using pulses with a pulse time not longer than the specified time.

Preferably, the excitation device is a pulsed laser, a pulsed light-emitting diode, or a combination of continuous light sources modulated in different ways.

Preferably, the excitation device and the detection device work synchronously.

Preferably, the excitation device includes one or more cylindrical mirrors, cylindrical lenses, deformable mirrors, transmission phase devices, and reflection phase devices to form the light sheet illumination.

Preferably, the excitation device generates at least one excitation beam approximately perpendicular to the detection direction of the detection device within the detection region.

Preferably, the excitation device comprises at least one scanning mechanism capable of moving the excitation beam in a scanning manner.

Preferably, the change of the beam radius of the excitation beam along the beam direction within the detection area does not exceed 3 times.

Preferably, the scanning mechanism includes a combination of one or more scanning galvanometers, resonant scanning mirrors, rotating polygonal mirrors, and acousto-optic modulators.

Preferably, the detection device uses a matrix photosensitive device as an imaging device, and the imaging device works synchronously with the scanning mechanism.

Preferably, the detection device includes a descanning mechanism that works synchronously with the scanning mechanism, and uses an array or matrix photosensitive device as an imaging device.

Preferably, the excitation device includes a beam splitting mechanism for splitting one excitation beam into multiple excitation beams.

Preferably, the beam splitting mechanism may include a combination of one or more lens arrays, mirror arrays, and half-mirror arrays, or include a fixed or adjustable phase filter or a digital micromirror array.

Preferably, the excitation device and the detection device form a scanning confocal imaging structure.

The present invention also proposes a method for three-dimensional microscopic imaging, including: using at least one excitation device to generate a detectable contrast in the sample area to be tested along the direction of the excitation axis; using at least one detection device to detect the sample along the direction of the detection axis For the contrast generated in the area to be measured, the detection device does not exclude the common components with the excitation device; wherein, the direction of relative motion between the sample, the excitation device and the detection device is neither parallel to the direction of the excitation axis nor perpendicular; the direction of the relative motion is neither parallel nor perpendicular to the direction of the principal axis of detection.

The present invention preferably uses methods such as deconvolution to eliminate blur caused by motion.

(3) Beneficial effects

According to the above device and method provided by the present invention, the advantages and beneficial effects of the present invention include: reducing imaging pauses in three-dimensional imaging, including uninterrupted imaging while the sample is moving, reducing field of view switching that must interrupt imaging, and eliminating Blur caused by sample motion during imaging improves aberrations caused in previous implementations. Compared with the prior art, the device and method provided by the invention can improve the throughput of three-dimensional imaging under the condition of the same resolution.

Description of drawings

Figures 1A to 1G show the solutions commonly used in the prior art to realize 3D imaging;

2A to 2C show the three-dimensional imaging principle of the present invention;

Fig. 3 shows the schematic diagram of a three-dimensional imaging microscope, as an application example of the present invention, the mark of each part in the accompanying drawing is as follows: 1, excitation device; 2, detection device; 3, sample stage; 4, excitation light source; 5. Scanning mirror; 6, 7. Relay lens; 8. Mirror; 9. Mirror or dichroic mirror; 10. Objective lens; 11. Excitation and detection area in the sample; 12. Objective lens; 13. Tube lens ; 14. Camera;

Figure 4 shows an example of mouse brain fluorescence imaging collected by the system shown in Figure 3, where 4(a) shows a three-dimensional reconstructed coronal section view of the imaging result of a brain slice with a thickness of hundreds of microns, and 4(b) shows The fine structure of neuron cell bodies and dendrites in a small area of the brain slice, 4(c) shows the fine structure of cells such as neuron axons captured in a certain imaging plane;

Fig. 5 has shown the schematic diagram of another three-dimensional imaging microscope, as another application example of the present invention, the mark of each part in the accompanying drawing is as follows: 1, excitation device; 2, detection device; 3, sample platform; 4, excite Light source; 5. Cylindrical mirror; 10. Objective lens; 11. Excitation and detection area in the sample; 12. Objective lens; 13. Tube lens; 14. Camera;

Fig. 6 has shown the schematic diagram of another three-dimensional imaging microscope, and as another application example of the present invention, the mark of each part in the accompanying drawing is as follows: 1, excitation device; 2, detection device; 3, sample platform; 4, excite Light source; 5. Two-dimensional scanning mechanism; 6, 7. Relay lens; 8. Mirror; 9. Dichroic mirror; 10. Objective lens; 11. Excitation and detection area in the sample; 12. Tube lens; 13. Camera ;

Figure 7 is an illustration of the deconvolution algorithm to eliminate motion blur to improve image quality, in which Figure 7A is the nano-fluorescent beads taken at rest; Figure 7B is the situation of 10 millisecond exposure when moving at 1 mm per second; The result after processing;

Fig. 8 illustrates the principle that the microscope and the imaging method of the present invention use a continuous light source to suppress motion blur through scanning;

FIG. 9 illustrates that the microscope and imaging method of the present invention use refractive index matching to improve the quality of images collected by the microscope of the present invention.

Detailed ways

In the following content of the invention, we will demonstrate a device and method that can not interrupt the imaging during the first motion and minimize the existence of the second motion, thereby increasing the effective imaging time and improving the imaging throughput.

The core content of the present invention is to reduce unnecessary interruption of imaging. A series of technology combinations are adopted around this content. The blur caused by sample movement during imaging improves the aberration caused by the aforementioned implementation, etc.

In order to solve the above technical problems, according to one aspect of the present invention, a microscope with three-dimensional imaging capability is provided, including at least one excitation device, which is used to generate detectable contrast in the sample area to be measured along the direction of the excitation axis. The microscope also includes at least one detection device, which is used to detect the contrast produced in the region of the sample to be measured along the direction of the detection axis, and the detection device does not exclude sharing elements with the excitation device; the microscope also includes at least one moving mechanism , for generating relative motion of the sample with the excitation means and the detection means. In the overall process of imaging a three-dimensional region of the sample, both the imaging and the relative motion have the characteristics of non-stop, and the direction of the relative motion is neither parallel nor perpendicular to the direction of the excitation principal axis and the detection principal axis. Here, the choice regarding the direction of relative motion helps to reduce field of view transitions that must interrupt imaging.

Since the relative movement during imaging has the characteristics of uniform speed and basically no pause, the irregular vibration caused by speed change or starting and stopping is eliminated. The blurring caused by motion that may still exist is simple and consistent, and can be easily eliminated by deconvolution and other methods.

Further, a technical solution that can be adopted in the present invention is that in the effective detection area of the detection device, the sample unit corresponding to each imaged voxel is only excited by the excitation device for no longer than a specific time. The displacement generated by the relative movement in time is not greater than the resolution requirement of the microscope. In this way, the relative motion produces negligible motion blur.

Furthermore, a technical solution that can be adopted in the present invention is to fill the environment where the sample is located with a transparent substance during imaging, and the transparent substance is approximately equal to the refractive index of the sample at that time.

The excitation device can use a combination of one or several light-emitting diodes, continuous lasers, and pulsed lasers as the excitation light source. The detectable contrast is one or a combination of signals such as fluorescence, elastic scattered light, Raman scattering, SHG, THG, and stimulated Raman scattering. The detection device uses one or a combination of CCD, CMOS photosensitive element, photodiode (PD) including avalanche photodiode (APD), photomultiplier tube, point, array or matrix device as the photosensitive device.

The excitation device can excite the sample with light sheet illumination using a pulse whose pulse time is not longer than the specified time, and the detection device uses a matrix photosensitive device such as a CCD or a CMOS camera as an imaging device.

The excitation device may include one or more cylindrical mirrors, cylindrical lenses, deformable mirrors, transmissive phase devices, and reflective phase devices to form the light sheet illumination.

The pulsed light source may comprise one or several pulsed lasers, pulsed LEDs or a combination of continuous light sources modulated in different ways. And the pulse light source can work synchronously with the imaging device.

One embodiment of the present invention is that the excitation device generates at least one excitation beam that is approximately perpendicular to the detection direction of the detection device; the excitation device includes at least one scanning mechanism, and the scanning mechanism is capable of scanning the excitation beam move.

Preferably, the change of the beam radius of the excitation beam along the beam direction within the detection area does not exceed 3 times.

One embodiment of the scanning mechanism is a combination of one or more scanning galvanometers (galvanometer scanners), resonant scanning mirrors, rotating polygonal mirrors, and acousto-optic modulators.

Another embodiment is that the detection device uses a matrix photosensitive device such as a CCD or a CMOS camera as an imaging device, and the imaging device works synchronously with the scanning mechanism. The detection device may include a descanning mechanism that works synchronously with the scanning mechanism, and an array or matrix photosensitive device such as an LED array, a CCD, or a CMOS camera is used as an imaging device.

According to the present invention, the excitation device may comprise a beam splitting mechanism for splitting one excitation beam into a plurality of excitation beams. The beam splitting mechanism may comprise a combination of one or more lens arrays, mirror arrays, half-mirror arrays, or a fixed or adjustable phase filter or a digital micromirror array (DMD).

One embodiment is that the excitation device and the detection device constitute a scanning confocal imaging structure.

In addition, the present invention also proposes to provide a three-dimensional microscopic imaging method, including: using at least one excitation device to generate a detectable contrast in the sample area to be measured along the excitation axis; using at least one detection device to generate a detectable contrast along the detection axis The direction detects the contrast produced in the region of the sample to be measured, and the detection device does not exclude sharing components with the excitation device; at least one moving mechanism is used to generate relative motion between the sample, the excitation device and the detection device. In the overall process of imaging a three-dimensional region of the sample, both the imaging and the relative motion have the characteristics of non-stop, and the direction of the relative motion is neither parallel nor perpendicular to the direction of the excitation principal axis and the detection principal axis.

2A to 2C show the three-dimensional imaging principle of the present invention. As shown in Figure 2A to Figure 2C, while imaging, the sample and the imaging system maintain a continuous motion at a constant speed, and the motion direction is neither parallel nor coincident with the imaging direction. Figure 2A shows that the motion direction and the imaging direction are at 45° Examples of included angles; as shown in FIG. 2B and FIG. 2C , the present invention continuously and uninterruptedly images new positions of a large-scale sample, and finally completes three-dimensional imaging of the sample.

Specific embodiments of the present invention are described below with reference to the accompanying drawings.

As an application example of the present invention, Fig. 3 shows a three-dimensional imaging microscope, for a sample with a thickness of 300 microns, the resolution requirement is 1 micron. The contrast that imaging depends on is exemplified by fluorescence. As an example, fluorescence contrast is provided by excitation of fluorescent proteins such as GFP with a 488 µm laser. The excitation device 1 and the detection device 2 may adopt a symmetrical multiplexing structure, that is, both sides include excitation and detection functions. If some devices in the shaded area are canceled, the left side is simplified as a simple excitation device, and the right side is a detection device. The principal axes of the optical paths of the excitation device 1 and the detection device 2 are vertical, and are at a 45° angle to the horizontal plane (desktop, ground). horn. The sample stage 3 is parallel to the horizontal plane, and is used to carry the sample and move the sample to the imaging position, and the sample moves horizontally continuously during imaging. The front end of the excitation device 1 and the detection device 2 is an objective lens 10 (Olympus UMPLFLN 20XW), with a numerical aperture of 0.5 and a working distance of 3.5 mm. The 488 micron laser is used as the excitation light 4 to be incident on the scanning galvanometer 5 through a telescope or aperture (not shown) whose intensity is adjusted and the beam diameter is adjusted, so as to generate linear excitation light that scans in a plane perpendicular to the detection direction after the objective lens 10 . The relay lenses 6 and 7 make the scanning galvanometer 5 conjugate to the rear focal plane of the objective lens 10 . The mirrors 8 and 9 are used to reverse the scanning line and save space; wherein, in a system including devices in the shaded region, the mirror 9 needs to be a dichroic mirror that passes long wavelengths. The excitation device 1 focuses the excitation beam on the position shown as 11 in the sample to excite fluorescence. The effective numerical aperture of the excitation device 1 is 0.03-0.04, so as to ensure that the waist radius of the concentrated excitation beam does not change more than twice in the range of about 420 microns near the thinnest point. The detection device 2 images the position shown in 11, and its core components are the objective lens 12, the lens tube lens 13 (125 mm focal length) and the camera 14 (Hamamatsu Flash4.0 sCMOS camera). The number of pixels of the camera is 2048×2048, the pixel size is 6.5×6.5 microns, and the full-frame frame rate is 100Hz, that is, each frame is 10 milliseconds. The vibrating mirror 5 scans synchronously with a one-way 10 millisecond sawtooth wave. The moving speed of the sample stage 3 is 100 microns/second, that is, 1 micron/frame. Motion-induced blur is less than 1/200 and can be ignored. Samples are matched to a refractive index of 1.45, and optional matching solutions include HistoDenz. It takes about 16 minutes for the system to complete three-dimensional imaging with a resolution of 1 micron for a 1×1 cm sample, and about 5-6 hours for mouse whole brain fluorescence imaging, which is significantly better than the prior art. Figures 4A to 4C show examples of mouse brain fluorescence imaging collected by the system, wherein 4A shows a three-dimensional reconstructed coronal view of a brain slice with a thickness of hundreds of microns, and Figure 4B shows a small brain slice. The fine structure of neuron cell bodies and dendrites in the range, Figure 4C shows the fine structure of cells such as neuron axons captured in a certain imaging plane.

In another embodiment, as shown in FIG. 5 , a structure similar to that of FIG. 3 is adopted but a pulsed light source is used to illuminate in the form of a light sheet. Among them, the pulse light source 4 is a nanosecond nitrogen molecule/dye laser with a working frequency of 30-100 Hz as an example. A telescope or diaphragm (not shown) with intensity and beam diameter adjustment forms a rectangular beam that enters the cylindrical mirror 5 . The focal length of the cylindrical mirror can be selected as 100 mm, and the cylindrical surface is perpendicular to the plane determined by the main axes of the optical paths of the excitation device 1 and the detection device 2 . The rest of the components are the same as the system shown in Figure 3. Camera image acquisition is performed simultaneously with the pulsed light source. When the operating frequency is 100Hz, the three-dimensional imaging capability of the system is the same as that of the system shown in Fig. 5 .

In yet another embodiment, as shown in FIG. 6 , a multi-photon scanning imaging structure is used. The excitation device 1 and the detection device 2 multiplex the front-end components. The main axis of the optical path of the exciting device 1 (and the detecting device 2) forms an angle of 45° with the vertical direction of the desktop (ground). The sample stage 3 is parallel to the desktop, carries the sample and moves the sample to the imaging position, and the sample moves horizontally continuously during imaging. The front end of the excitation device 1 and the detection device 2 is an objective lens 10 (Olympus UMPLFLN 20XW), with a numerical aperture of 0.5 and a working distance of 3.5 mm. The pulsed excitation light 4 that can generate multiphoton excitation, usually a femtosecond or picosecond laser, is incident on a two-dimensional scanning mechanism 5 through a telescope or diaphragm (not shown) with intensity adjustment and beam diameter adjustment, to generate Point-like excitation scanned in a plane perpendicular to the probing direction. The relay lenses 6 and 7 make the vibrating mirror 5 conjugate to the rear focal plane of the objective lens 10 . The mirrors 8 and 9 are used to reverse the scanning line and save space; wherein, the mirror 9 is a dichroic mirror that passes long wavelengths. The effective numerical aperture of the excitation device 1 is 0.5. The excitation device 1 generates excitation at the position shown as 11 in the sample. The detection device 2 images the position shown in 11 , and its core components are similar to the system shown in FIG. 5 . The system can be applied in situations where the signal-to-noise ratio is required higher than the speed.

In yet another embodiment, in a device similar to the system shown in FIG. 3 or FIG. 6 , a beam splitting mechanism may be inserted before the excitation light 4 enters the scanning mechanism 5 for splitting an excitation beam into multiple excitation beams. The light beam excites different sub-regions within the range of the imaging position 11 at the same time, thereby speeding up the overall scanning speed.

In yet another embodiment, similar to the system shown in Figure 6, as well known to experts in the field of microscopic imaging, its detection device can use other types of high-speed detectors, such as photodiodes (PDs) including avalanche photodiodes (APDs). , photomultiplier tube, etc.

In yet another embodiment, similar to the system shown in FIG. 6 , as is well known to experts in the field of microscopic imaging, the multiplexed front-end devices of the excitation device 1 and the detection device 2 may include tracing from the objective lens to the scanning mechanism, and then A confocal pinhole is installed at the position conjugated with the sample in the separated detection part, thereby forming a confocal scanning imaging structure.

In another embodiment, similar to the aforementioned systems, as well known to experts in the field of microscopic imaging, the excitation light source used by the excitation device can be a combination of one or several light-emitting diodes, continuous lasers, and pulsed lasers; The contrast can be one or a combination of fluorescence, elastic scattered light, Raman scattering, SHG, THG, stimulated Raman scattering and other signals.

In the above-mentioned embodiments, only the case where the detection direction forms an included angle of 45° with the relative motion direction is exemplified. In other applications, the included angle can be flexibly selected according to requirements, mainly adapting to the selected contact angle of the objective lens. For example, an objective lens with a larger numerical aperture may be selected for a system with a higher resolution requirement, and accordingly, the included angle may be selected to be 55°-65°. Likewise, the form of relative motion is not limited to a straight line. For example, a circular motion may be another preferred form.

Due to motion during imaging, the captured image will have motion-induced blur. Since the motion pattern here is simple and fixed, as a solution, the present invention can use a conventional motion blur removal algorithm to effectively improve the quality of the captured image. Figure 7A is the nano fluorescent beads taken at rest; Figure 7B is the situation of 10 millisecond exposure when moving at a speed of 1 mm per second, it can be seen from the figure that there is obvious but consistent motion blur; deconvolution processing can The image details are effectively recovered, and the results are shown in Fig. 7C. As another solution, the method of limiting the excitation time can also be used to suppress motion blur. The main point is to ensure that the relative resolution of the motion of each point in the sample within the excitation time can be ignored. Simply put, the excitation time is multiplied by the motion The speed is significantly smaller than the minimum resolution scale. If the resolution requirement is 1 micron and the motion speed is 1 mm/s, the excitation time is required to be less than 1 millisecond. A pulsed light source with an appropriate pulse width can directly meet this requirement. CW light sources can also be modulated by appropriate switches for lighting, but more efficiently, CW light sources can also be scanned to achieve short-term excitation of points. As shown in Figure 8 as an example, the field of view of the microscope, taking the imaging camera as a reference, has a width of 2000 pixels. Correspondingly, the width of the line-shaped excitation light is about 10 pixels. When the excitation light sweeps across the entire field of view within 0.01 second, the actual time for each pixel to be excited is only about 0.01 second divided by 2000 times 10 or 50 microseconds.

On the other hand, if there is a difference in the refractive index of the space where the sample is located, including the paths that the excitation light leaves the excitation device to enter the sample and the path that the signal light passes from the sample to the detection device, the thicker sample may lose its transparency, and at the same time, there may be a larger imaging gap. aberrations. To obtain a good signal-to-noise ratio and resolution, the present invention proposes a clearing/refractive index normalization process in the approach. Figure 9 shows the effect comparison before and after treatment.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (20)

1. A microscope with three-dimensional imaging capability, comprising: at least one excitation device, used to generate a detectable contrast in the region of the sample to be measured along the direction of the excitation axis; at least one detection device, used to detect the sample along the direction of the detection axis the contrast generated in the area to be measured; at least one movement mechanism for generating relative movement of the sample and said excitation means and detection means; Wherein, the direction of the relative movement is neither parallel nor perpendicular to the direction of the excitation principal axis; the direction of the relative movement is neither parallel nor perpendicular to the direction of the detection principal axis. 2. The microscope with three-dimensional imaging capability as claimed in claim 1, characterized in that, in the effective detection area of the detection device, the sample unit corresponding to each voxel of imaging is detected only within a specified time The excitation device is excited, and the displacement generated by the relative movement within the specified time is not greater than the resolution requirement of the microscope. 3. The microscope with three-dimensional imaging capability according to claim 1 or 2, characterized in that the environment where the sample is located is filled with a transparent substance, and the transparent substance is approximately equal to the refractive index of the sample at that time. 4. The microscope with three-dimensional imaging capability according to claim 1, characterized in that, the excitation device excites the light source with a combination of one or several light emitting diodes, continuous lasers, and pulsed lasers. 5. The microscope with three-dimensional imaging capability as claimed in claim 1, wherein the detectable contrast is the signal of fluorescence, elastic scattered light, Raman scattering, SHG, THG, stimulated Raman scattering, etc. one or a combination. 6 . The microscope with three-dimensional imaging capability according to claim 1 , wherein the excitation device excites the sample by means of light sheet illumination using a pulse whose pulse time is not longer than the specified time. 7. The microscope with three-dimensional imaging capability according to claim 6, wherein the exciting device is a pulsed laser, a pulsed light emitting diode, or a combination of continuous light sources modulated by different methods. 8. The microscope with three-dimensional imaging capability according to claim 6, characterized in that, the excitation device and the detection device work synchronously. 9. The microscope with three-dimensional imaging capability as claimed in claim 6, wherein the excitation device comprises one or more cylindrical mirrors, cylindrical lenses, deformable mirrors, transmission phase devices, reflection phase devices, for The light sheet illumination is formed. 10 . The microscope with three-dimensional imaging capability according to claim 1 , wherein the excitation device generates at least one excitation beam approximately perpendicular to the detection direction of the detection device within the detection area. 11 . 11. The microscope with three-dimensional imaging capability according to claim 10, wherein the excitation device comprises at least one scanning mechanism capable of scanning and moving the excitation beam. 12 . The microscope with three-dimensional imaging capability according to claim 11 , wherein the change of the beam radius of the excitation beam along the beam direction within the detection area is no more than 3 times. 13 . 13. The microscope with three-dimensional imaging capability according to claim 11, wherein the scanning mechanism comprises a combination of one or more scanning galvanometers, resonant scanning mirrors, rotating polygonal mirrors, and acousto-optic modulators. 14. The microscope with three-dimensional imaging capability according to claim 11, characterized in that, the detection device uses a matrix photosensitive device as an imaging device, and the imaging device works synchronously with the scanning mechanism. 15. The microscope with three-dimensional imaging capability according to claim 11, characterized in that, the detection device includes a descanning mechanism that works synchronously with the scanning mechanism, and an array or matrix photosensitive device is used as an imaging device. 16. The microscope with three-dimensional imaging capability according to claim 1, wherein the excitation device comprises a beam splitting mechanism for splitting one excitation beam into multiple excitation beams. 17. The microscope with three-dimensional imaging capability as claimed in claim 16, wherein the beam splitting mechanism may comprise a combination of one or more lens arrays, reflector arrays, and half-mirror arrays, or include fixed Or a tunable phase filter or a digital micromirror array. 18. The microscope with three-dimensional imaging capability according to claim 1, characterized in that, the excitation device and the detection device constitute a scanning confocal imaging structure. 19. A method for three-dimensional microscopic imaging, comprising: Using at least one excitation device to generate a detectable contrast in the region of the sample to be measured along the direction of the excitation axis; Using at least one detection device to detect the contrast generated in the region of the sample to be measured along the direction of the detection axis, the detection device does not exclude sharing elements with the excitation device; wherein, The direction of the relative movement of the sample, the excitation device and the detection device is neither parallel nor perpendicular to the direction of the excitation principal axis; the direction of the relative movement is neither parallel nor perpendicular to the direction of the detection principal axis. 20. The method for three-dimensional microscopic imaging according to claim 19, further comprising: eliminating the blur caused by the relative motion by using methods such as deconvolution.